The present invention defines a new use for the known cancer antigen PRAME (PReferentially expressed Antigen in MElanoma), and in particular provides methods for the stratification of cancer patients based on PRAME expression and for the treatment of subjects with cancer by the inhibition of PRAME expression or activity.
Retinoic acid (RA) induces proliferation arrest, differentiation and apoptosis in a wide variety of cell types (Altucci, 2001; Freemantle, 2003). Defects in retinoic acid receptor (RAR) signalling, such as those caused by the RML-RARα and PLZF-RARα translocations in acute promyelocytic leukaemia, have been implicated in cancer (Altucci, 2001; Freemantle, 2003. Binding of RA to its receptor leads to release of co-repressor molecules and recruitment of co-activators to RAR, resulting in activation of transcription (Xu, 1999).
Acetylation and deacetylation of histones is associated with transcriptional events leading to cell proliferation and/or differentiation. Regulation of the function of transcription factors is also mediated through acetylation. Recent reviews of histone deacetylation include Kouzarides, 1999 and Pazin et al., 1997.
The correlation between the acetylation status of histones and the transcription of genes has been known for over 30 years (see, for example, Howe et al., 1999). Certain enzymes, specifically acetylases (e.g., histone acetyltransferase, HAT) and deacetylases (e.g., histone deacetylase, HDAC), which regulate the acetylation state of histones have been identified in many organisms and have been implicated in the regulation of numerous genes, confirming the link between acetylation and transcription. See, for example, Davie, 1998. In general, histone acetylation correlates with transcriptional activation, whereas histone deacetylation is associated with gene repression.
A growing number of histone deacetylases (HDACs) have been identified (see, for example, Ng and Bird, 2000). The first deacetylase, HDAC1, was identified in 1996 (see, for example, Tauton et al., 1996). Subsequently, two other nuclear mammalian deacetylases were found, HDAC2 and HDAC3 (see, for example, Yang et al., 1996, 1997, and Emiliani et al., 1998). See also, Grozinger et al., 1999; Kao et al., 2000; and Van den Wyngaert et al., 2000.
HDACs function as part of large multiprotein complexes, which are tethered to the promoter and repress transcription. Well characterised transcriptional repressors such as Mad (Laherty et al., 1997), pRb (Brehm et al., 1998), nuclear receptors (Wong et al., 1998) and YY1 (Yang et al., 1997) associate with HDAC complexes to exert their repressor function.
The study of inhibitors of histone deacetylases indicates that these enzymes play an important role in cell proliferation and differentiation. The inhibitor Trichostatin A (TSA) (Yoshida et al., 1990a) causes cell cycle arrest at both G1 and G2 phases (Yoshida and Beppu, 1988), reverts the transformed phenotype of different cell lines, and induces differentiation of Friend leukaemia cells and others (Yoshida et al., 1990b). TSA (and SAHA) have been reported to inhibit cell growth, induce terminal differentiation, and prevent the formation of tumours in mice (Finnin et al., 1999).
The activity of HDACs and HATS (Histone Acetyl Transferases) is frequently deregulated in cancer and one of the ways in which these enzymes are involved in cancer is in the repression of retinoic acid receptor signalling.
The clear involvement of HDACs in the control of cell proliferation and differentiation suggests that aberrant HDAC activity may play a role in cancer. The most direct demonstration that deacetylases contribute to cancer development comes from the analysis of different acute promyelocytic leukemias (APL). In most APL patients, a translocation of chromosomes 15 and 17 (t(15;17)) results in the expression of a fusion protein containing the N-terminal portion of PML gene product linked to most of RARα (retinoic acid receptor). In some cases, a different translocation (t(11;17)) causes the fusion between the zinc finger protein PLZF and RARα. In the absence of ligand, the wild type RARα represses target genes by tethering HDAC repressor complexes to the promoter DNA. During normal hematopoiesis, retinoic acid (RA) binds RARα and displaces the repressor complex, allowing expression of genes implicated in myeloid differentiation, The RARα fusion proteins occurring in APL patients are no longer responsive to physiological levels of RA and they interfere with the expression of the RA-inducible genes that promote myeloid differentiation. This results in a clonal expansion of promyelocytic cells and development of leukaemia. In vitro experiments have shown that TSA is capable of restoring RA-responsiveness to the fusion RARα proteins and of allowing myeloid differentiation. These results establish a link between HDACs and oncogenesis and suggest that HDACs are potential targets for pharmaceutical intervention in APL patients. (See, for example, Kitamura et al., 2000; David et al., 1998; Lin et al., 1998).
Furthermore, different lines of evidence suggest that HDACs may be important therapeutic targets in other types of cancer. Cell lines derived from many different cancers (prostate, colorectal, breast, neuronal, hepatic) are induced to differentiate by HDAC inhibitors (Yoshida and Horinouchi, 1999). A number of HDAC inhibitors have been studied in animal models of cancer. They reduce tumour growth and prolong the lifespan of mice bearing different types of transplanted tumours, including melanoma, leukaemia, colon, lung and gastric carcinomas, etc. (Ueda et al., 1994; Kim et al., 1999).
Thus, although HDAC inhibitors (HDACi) are a promising new class of anti-cancer drug, the molecular basis for their selective growth-inhibitory activity on cancer cells is at present unclear.
PRAME was first identified as an antigen in human melanoma that triggers cytotoxic T cell-mediated anti-tumour immune responses (Ikeda et al, 1997). PRAME is also over-expressed in a variety of other human malignancies, including acute and chronic leukemias, non-small-cell lung carcinoma, head and neck cancer, renal carcinoma, and its expression is prognostic for a poor clinical outcome in breast cancer (Ikeda, 1997; van't Veer 2002; van Hazen, 1998; Neumann 1998; Boon, 2003). However, no function for PRAME has been described to date.
The present invention demonstrates that PRAME expression inhibits retinoic acid-induced differentiation, growth arrest and apoptosis and that PRAME is, therefore, a dominant repressor, or negative regulator, of RAR signalling. The invention also shows that PRAME suppresses the HDACi-mediated activation of RAR signalling. These discoveries of a function for PRAME have opened up a new avenue for the treatment of cancer, via the suppression of PRAME, as well as enabling the stratification of subjects prior to treatment with known anti-cancer treatments, such as retinoids and HDACi's.